Journal Article

Loss of DNA–protein crosslinks from formaldehyde-exposed cells occurs through spontaneous hydrolysis and an active repair process linked to proteosome function

George Quievryn and Anatoly Zhitkovich

in Carcinogenesis

Volume 21, issue 8, pages 1573-1580
Published in print August 2000 | ISSN: 0143-3334
Published online August 2000 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/21.8.1573
Loss of DNA–protein crosslinks from formaldehyde-exposed cells occurs through spontaneous hydrolysis and an active repair process linked to proteosome function

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DNA–protein crosslinks (DPC) involving all major histones are the dominant form of DNA damage in formaldehyde-exposed cells. In order to understand the repair mechanisms for these lesions we conducted detailed analysis of the stability of formaldehyde-induced DPC in vitro and in human cells. DNA–histone linkages were found to be hydrolytically unstable, with t½ = 18.3 h at 37°C. When histones were allowed to remain bound to DNA after crosslink breakage, the half-life of DPC increased to 26.3 h. This suggests that ~30% of spontaneously broken DPC could be re-established under physiological conditions. The half-lives of DPC in three human cell lines (HF/SV fibroblasts, kidney Ad293 and lung A549 cells) were similar and averaged 12.5 h (range 11.6–13.0 h). After adjustment for spontaneous loss, an active repair process was calculated to eliminate DPC from these cells with an average t½ = 23.3 h. Removal of DPC from peripheral human lymphocytes was slower (t½ = 18.1 h), due to inefficient active repair (t½ = 66.6 h). This indicates that the major portion of DPC is lost from lymphocytes through spontaneous hydrolysis rather than being actively repaired. Depletion of intracellular glutathione from A549 cells had no significant effect on the initial levels of DPC, the rate of their repair or cell survival. Nucleotide excision repair does not appear to be involved in the removal of DPC, since the kinetics of DPC elimination in XP-A and XP-F fibroblasts were very similar to normal cells. Incubation of normal or XP-A cells with lactacystin, a specific inhibitor of proteosomes, caused inhibition of DPC repair, suggesting that the active removal of DPC in cells may involve proteolytic degradation of crosslinked proteins. XP-F cells showed somewhat higher sensitivity to formaldehyde, possibly signaling participation of XPF protein in the removal of residual peptide–DNA adducts.

Keywords: BSA, bovine serum albumin; BSO, l-buthionine-R,S-sulfoximine; DPC, DNA–protein crosslinks; DPTA, diethylenetriaminepentaacetic acid; FA, formaldehyde; GSH, glutathione; mBBr, monobromobimane; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; XP, Xeroderma pigmentosum.

Journal Article.  6224 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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